Acta Biologica Hungarica 59 (3), pp. 365–383 (2008) DOI: 10.1556/ABiol.59.2008.3.10 MOLECULAR PHYLOGENETIC AND SCANNING ELECTRON MICROSCOPICAL ANALYSES PLACES THE CHOANEPHORACEAE AND THE GILBERTELLACEAE IN A MONOPHYLETIC GROUP WITHIN THE MUCORALES (ZYGOMYCETES, FUNGI) KERSTIN VOIGT1* and L. OLSSON2 1 Institut für Mikrobiologie, Pilz-Referenz-Zentrum, Friedrich-Schiller-Universität Jena, Neugasse 24, D-07743 Jena, Germany 2 Institut für Spezielle Zoologie und Evolutionsbiologie, Friedrich-Schiller-Universität Jena, Erbertstr. 1, D-07743 Jena, Germany (Received: May 4, 2007; accepted: June 11, 2007) A multi-gene genealogy based on maximum parsimony and distance analyses of the exonic genes for actin (act) and translation elongation factor 1 alpha (tef ), the nuclear genes for the small (18S) and large (28S) subunit ribosomal RNA (comprising 807, 1092, 1863, 389 characters, respectively) of all 50 gen- era of the Mucorales (Zygomycetes) suggests that the Choanephoraceae is a monophyletic group. The monotypic Gilbertellaceae appears in close phylogenetic relatedness to the Choanephoraceae. The mono- phyly of the Choanephoraceae has moderate to strong support (bootstrap proportions 67% and 96% in distance and maximum parsimony analyses, respectively), whereas the monophyly of the Choanephoraceae-Gilbertellaceae clade is supported by high bootstrap values (100% and 98%). This suggests that the two families can be joined into one family, which leads to the elimination of the Gilbertellaceae as a separate family. In order to test this hypothesis single-locus neighbor-joining analy- ses were performed on nuclear genes of the 18S, 5.8S, 28S and internal transcribed spacer (ITS) 1 ribo- somal RNA and the translation elongation factor 1 alpha (tef ) and beta tubulin (βtub) nucleotide sequences. The common monophyletic origin of the Choanephoraceae-Gilbertellaceae clade could be confirmed in all gene trees and by investigation of their ultrastructure. Sporangia with persistent, sutured walls splitting in half at maturity and ellipsoidal sporangiospores with striated ornamentations and polar ciliate appendages arising from spores in persistent sporangia and dehiscent sporangiola represent synapomorphic characters of this group. We discuss our data in the context of the historical development of their taxonomy and physiology and propose a reduction of the two families to one family, the Choanephoraceae sensu lato comprising species which are facultative plant pathogens and parasites, especially in subtropical to tropical regions. Keywords: Multigene genealogy – ultrastructure – ITS – nrDNA – protein-coding genes * Corresponding author; e-mail: [email protected] 0236-5383/$ 20.00 © 2008 Akadémiai Kiadó, Budapest 366 KERSTIN VOIGT and L. OLSSON INTRODUCTION The Choanephoraceae J. Schröter 1894 consists of fungi parasitic on higher plants causing blossom blights and fruit rots [13, 33, 35]. They are geographically wide- spread, especially in subtropical and tropical regions. Systematically, the Choanephoraceae is one of thirteen families of the Mucorales (Zygomycetes) recog- nized in the current classification scheme [17, 18]. The Choanephoraceae comprises three genera, Choanephora Thaxter 1903 (originally: Cunninghamia Currey 1873), Blakeslea Thaxter 1914 and Poitrasia P. M. Kirk 1984 with a total of five species, which are characterized by formation of large, pyriform collumellate, Mucor-like, multi-spored sporangia and smaller, non-collumellate, few- and uni-spored, dehis- cent or non-dehiscent sporangiola borne on separate and distinct sporophores or spo- rangiophores [17]. The sporangiospores from sporangia and sporangiola are mor- phologically similar, namely ellipsoidal in shape, pigmented, longitudinally striated and possessing clusters of radiating hyaline appendages at both of their polar ends in sporangia and dehiscent sporangiola [1, 13, 23, 28, 43]. Zygospore morphology appears to be unique for the Choanephoraceae and is proposed to be significant as a family-specific criterion [1, 15–17]. The zygospores are at maturity smooth-walled, golden to dark brown, delicately striated and arise from the fusion of nearly equal- sized gametangia, which are developed by apposed or tong-like suspensors between the tips of basally entwined hyphal branches. During the past century the genus delimitation of the Choanephoraceae was sub- ject to constant changes. The genera are distinguished primarily by a combination of different sporulating structures, which forced different authors to include different genera into the family. Naumov [22] included only Choanephora and Blakeslea in the Choanephoraceae. Shanor et al. [32] described a new genus Cokeromyces Shanor 1950 and included it in the Choanephoraceae because of the way in which it repro- duces asexually (by formation of sporangiola on a capitate vesicle on erect sporan- giophores), which is similar to Blakeslea and Choanephora. The presence of few- and uni-spored sporangiola developing on spherical or longitudinal vesicles of the erect sporangiophore apex was considered to be important. This resulted in the pro- posal of a polyphyletic concept for the family Choanephoraceae uniting Blakeslea, Choanephora, Cunninghamella Matruchot 1903, Rhopalomyces Corda 1839, Radiomyces Embree 1959, Mycotypha Fenner 1932, Thamnocephalis Blakeslee 1905 and Sigmoideomyces Thaxter 1891 [43]. The genus Gilbertella Hesseltine 1960, which is based on a species isolated from a peach (Prunus persica) exhibiting storage rot, was originally included in the genus Choanephora as C. persicaria Eddy 1925 and was therefore part of the Choanephoraceae [11]. With the introduction of Gilbertella for Choanephora persicaria and Gilbertella’s separation from the genus Choanephora, which is based on the formation of non-choanephoraceous Mucor- type rough-walled, appendaged zygospores formed between two opposed suspen- sors, Hesseltine [12] placed Gilbertella in the Mucoraceae, a view shared by Zycha et al. [43], von Arx [1] and Kirk [16]. But Hesseltine and Ellis [14] allied Gilbertella again with Choanephora and Blakeslea in the Choanephoraceae. Moreover, other Acta Biologica Hungarica 59, 2008 Revision of the families Choanephoraceae and Gilbertellaceae 367 authors treated Gilbertella and Blakeslea as synonyms of Choanephora [19, 20]. Large-scale phylogenetic studies confirmed the close phylogenetic relatedness be- tween Gilbertella and the Choanephoraceae foreshadowing the findings of the pre- sent study [25, 40]. These contradictory views and interpretations show the importance of characters that are easy to define, restricted to a few taxa, and taxon-specific. The aim of the present study is to investigate members of the Choanephoraceae using molecular phylogenetics and scanning electron microscopy, and to prove the merging of the families Choanephoraceae and Gilbertellaceae Benny 1991. MATERIAL AND METHODS Fungal strains, media and growth conditions Eight fungal strains of the Choanephoraceae and the Gilbertellaceae were investigat- ed in this study: Blakeslea trispora FSU 331 and FSU 332, Choanephora infundibu- lifera f. cucurbitarum-FSU 314 and FSU 772, Gilbertella persicaria FSU 807 and FSU 808, Poitrasia circinans FSU 888 and FSU 889 (Table 1). Mucor hiemalis f. corticola FSU 3008, which was kindly provided by Prof. George Newcombe (University of Idaho, Moscow, Id, USA), was used as the outgroup taxon for spo- rangial ultrastructure comparison. All strains are deposited and maintained in the Fungal Reference Centre Jena and available upon request. The fungi were cultivated on MEX medium containing 30 g L–1 malt extract (Serva, Heidelberg, Germany) or on pea glucose medium consisting of 500 mL L–1 pea extract and 5 g L–1 glucose (Merck, Darmstadt, Germany). For pea extract preparation 150 g frozen peas were boiled in 500 mL distilled water for 15 min. For solidification the media were sup- plemented with 20 g L–1 agar agar (Roth, Karlsruhe, Germany). Petri dishes were incubated at 25 °C until sporulation for about 3–6 days. The fungal spores served as inoculum for the mycelial growth for DNA extraction procedures. Liquid cultures appropriate for each of the fungal strains were inoculated with spore suspensions (approximately 106 spores ml–1) in 500 ml round flasks. The liquid cultures were incubated for two to four days at 25 °C under constant shaking (120 rpm). Purification of genomic DNA and PCR amplification Genomic DNA was purified and amplified according to Einax and Voigt [7]. In typ- ical PCR experiments one assay contained 50–100 ng genomic DNA, 10 pmol for- ward primer F-βtub1 (5´-CARGCYGGTCARTGYGGTAACCA-3´), 10 pmol reverse primer F-βtub4r (5´-GCCTCAGTRAAYTCCATYTCRTCCAT-3´), 16.0 mM (NH4)2SO4, 50.0 mM Tris-HCl pH 8.8., 0.01% (v/v) Tween 20, 2.0 mM mag- nesium chloride, 0.2 mM of each dNTP (Roth, Karlsruhe, Germany) and 1 unit Taq polymerase (InViTek, Berlin, Germany) in a total reaction volume of 50 μl. After an Acta Biologica Hungarica 59, 2008 368 KERSTIN VOIGT and L. OLSSON Table 1 Strains analysed by scanning electron microscopy Mating Species and strain Equivalent strain designations type Blakeslea trispora FSU 331 + CBS 130.59, ATCC 14271, NRRL 2456 Blakeslea trispora FSU 332 – CBS 131.59, ATCC 14272, IMI 195169, NRRL 2457 Choanephora infundibulifera f. cucurbitarum + CBS 150.51 FSU 314 Choanephora infundibulifera f. cucurbitarum Nd DSM 960 FSU 772 Gilbertella persicaria var. indica FSU 807 + CBS 349.64 Gilbertella persicaria var. indica FSU 808 – CBS 442.64 Poitrasia circinans FSU 888 + CBS